Methods of treating outer eye disorders using high orp acid water and compositions thereof

ABSTRACT

The present invention relates to a method of treating an outer eye disorder selected from a cataract, neovascularization, keratitis, epithelium deficiency, or chronic opacity, by administering to the eye a composition comprising acidic electrolytic water. The present invention also relates to a stable acidic electrolyzed oxidizing water characterized by low conductivity, the presence of dissolved chlorine gas (Cl 2 ), hypochlorous acid (HOCl) and chloride ions (Cl − ), and by the presence of negligible quantities of hypochlorite ion (OCl − ).

RELATION TO PRIOR APPLICATIONS

This application claims priority to U.S. Provisional App. Nos.61/187,900 filed Jun. 17, 2009, and 61/239,912, filed Sep. 4, 2009. Thecontent of these earlier applications is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to pharmacological methods of treatingouter eye disorders using acid water having a high oxidation reductionpotential (ORP) and compositions thereof.

BACKGROUND OF THE INVENTION Outer Eye Disorders

The outer eye includes several organs, including the cornea, the irisand the lens. The iris is a membrane in the eye, responsible forcontrolling the amount of light reaching the retina. The cornea and lensrefract light onto the retina. Healthy eyes produce clear vision as theresult of the transparency of the cornea and lens. Cataract, a cloudingof the lens of the eye, can obstruct the passage of light and result ina gradual loss of vision. Unfortunately, very few treatments exist forcataract, other than to replace the cataractous lens with an artificiallens through a complicated surgical procedure.

Disorders such as keratitis, neovascularization, and epitheliumdeficiency can also reduce vision by interfering with the transparencyof the cornea. These disorders can result from numerous causes,including viral and bacterial infections, trauma and surgery.Antibiotics and anti-viral agents are often used to treat infectiouscauses, but in many instances the patient has no choice but to undergo acomplicated surgical procedure to remove damaged tissue before it canscar and reduce eyesight.

PCT Publications WO 2004/012748 and WO 2001/054704 teach an isotonicionized acidic solution for wound care, and tout the water based uponits antioxidant characteristics and antimicrobial properties. Thepublications state that the solution may be used in the place of salinein ophthalmic applications such as contact lens cleaning solutions orfor irrigation of the eye during ophthalmic surgery, and that theproperties of the solution depend on the particular concentration rangesof a mixture of salts.

There remains a need for pharmacological methods of treating outer eyedisorders, especially those that affect the cornea and lens. There isparticularly a need to methods that prevent further deterioration ofvision in the eye, and that potentially improve the vision of thepatient whose vision has worsened.

High ORP Acid Water

It is known that aqueous solutions of salts, particularly sodiumchloride, as a consequence of an electrolytic treatment, are split intotwo liquid products, one having basic and reducing characteristics(generally known as cathode water or alkaline water) and another(generally known as anode water or acid water) having acid and oxidizingcharacteristics.

Conventional electrolytic waters suffer the acknowledged drawback ofhaving very limited preservation. A few days after preparation, theproduct in fact generally tends to degrade and lose its properties.Known electrolytic waters, therefore, must be prepared and usedsubstantially on the spot. Accordingly, the commercial utilization ofthe product in itself is extremely disadvantageous, since the shelf lifeof any ready-made packages is dramatically limited.

The stability of an electrolyzed oxidizing water is reported in thearticle “Effects of Storage Conditions and pH on Chlorine Loss inElectrolyzed Oxidizing (EO) Water”—Journal of Agricultural and FoodChemistry—2002, 50, 209-212 by Soo-Voo Len, et al. In Soo-Voo Len,electrolyzed water with an acidic pH (2.5-2.6), high OPR (1020-1120 mV),and a free chlorine content of ˜50 ppm (53-56 ppm) was generated using acurrent intensity of 14 Ampere and 7.4 Volt. Unfortunately, in an opencondition at 25° C., the chlorine in the electrolyzed water wascompletely lost after 30 hours when agitated, and after 100 hours whennot agitated. Furthermore, in a closed dark condition at 25° C., thefree chlorine in the electrolyzed water decreased by approximately 40%after 1400 hours (about 2 months).

The stability of electrolyzed oxidizing water also is reported in thearticle “Effects of storage conditions on chemical and physicalproperties of electrolyzed oxidizing water”—Journal of Food Engineering65 (2004) 465-471 by Shun-Yao Hsu, et al. In Shun-Yao Hsu, theelectrolyzed water of “formulation J” had an acidic pH (2.61), high OPR(1147 mV), and a free chlorine content of 56 ppm. The article reportsthat in a closed condition at 25-30° C., the free chlorine in theelectrolyzed water was 43 ppm after 21 days, a 23% loss.

Thus, there remains a need for acidic electrolytic water with a greaterchemical stability than traditional waters. There is a particular needfor water with a greater stability during long term storage, so as toallow for the commercial utilization of acidic electrolytic waterproducts.

SUMMARY OF THE INVENTION

It has unexpectedly been discovered that acidic water, having a highoxidation reduction potential, promotes and facilitates an orderedregeneration of the eye when impacted by negative environmental stimulisuch as infections and trauma, or metabolic processes such as aging anddiabetes. In particular, it has been discovered that the water fosters ahealthy epithelium on the cornea, reduces uncontrolled cornealneovascularization, and encourages ordered protein synthesis in thelens. The water can thus be used to treat various outer eye disordersthat affect the cornea, lens or iris, including cataract, keratitis,neovascularization and epithelium deficiency.

Therefore, in one embodiment, the invention provides a method oftreating an outer eye disorder selected from cataract, keratitis,corneal neovascularization and epithelium deficiency in an animalpatient in need thereof, comprising topically administering to an eye ofsaid patient a composition comprising a therapeutically effective amountof a high ORP acidic water. The composition is preferably in the form ofan eye drop. In another embodiment, the invention provides a method forimproving opacity in a lens of an animal patient in need thereof,comprising administering to an eye of said patient a compositioncomprising a therapeutically effective amount of a high ORP acidicwater.

The methods can be performed ad libitum in response to observableirritation. An effective treatment for these disorders typicallyrequires a plurality of administrations, extending days, months or evenyears of the patient's life. The water can be defined by severalcharacteristics, including pH and ORP, in addition to othercharacteristics including cluster size (as measured by NMR half linewidth), and the content of various chlorine/chloride species.

It also has unexpectedly been discovered that acidic nanoclustered waterhaving a particular composition of chlorine species has a greaterchemical stability than traditional waters. The unique composition canresult from particular membrane and electrodes used in the electrolyzingequipment, which can produce a high current intensity without causingthe electrodes to break up on their surface and release heavy metalsthat may adversely affect stability.

Additional embodiments and advantages of the invention will be set forthin part in the description which follows, and in part will be obviousfrom the description, or may be learned by practice of the invention.The embodiments and advantages of the invention will be realized andattained by means of the elements and combinations particularly pointedout in the appended claims. It is to be understood that both theforegoing general description and the following detailed description areexemplary and explanatory only and are not restrictive of the invention,as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and together with the description, serve to explain theprinciples of the invention.

FIG. 1 is a schematic view of an electrolytic device comprising anelectrolysis chamber and two electrodes.

FIG. 2 is side cross-view of a human eye, depicting the variouscomponents of the eye.

FIG. 3 is a graph illustrating the modulation of the immune systemcytokines by Acidic Nanoclustered Water.

FIG. 4 is a set of graphs illustrating the concentration of variouschlorine species as a function of pH.

FIG. 5 is a graph illustrating the loss of free chlorine in high and lowchloride Acidic Nanoclustered Water in an open, agitated, exposedcondition over 24 hours.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing definitions and detailed description of preferred embodimentsof the invention and the non-limiting Examples included therein.

Definitions and Use of Terms

“Pharmaceutically acceptable” means that which is useful in preparing apharmaceutical composition that is generally safe, non-toxic and neitherbiologically nor otherwise undesirable and includes that which isacceptable for veterinary use as well as human pharmaceutical use.

As used herein, the term “fluid” is used to reference any pure fluid,solution or suspension which is capable of producing a non-spontaneouschemical reaction if subjected to electrolysis. One highly preferredfluid is water. The term “water” is used to reference any type of water,such as tap water, filtered water, deionized water, and distilled water.Once subjected to electrolysis, the water separates into two liquidfractions, which for the sake of simplicity are referenced here as acidwater or anode water and as cathode water or alkaline water.

The term “high ORP water” refers to water having an oxidation reductionpotential greater that +600. The ORP preferably ranges from +600 to+1350 mV, more preferably from +800, +900, or +1000 mV to +1300 mV, mostpreferably from +1100 to +1250 mV.

The term “acid water” or “acidic water” refers to water having a pH lessthan 7.0. The pH of the acid water preferably ranges from 0.5, 1.0 or2.0 to 6.5, 6.0, 5.0, 4.0, or 3.0, and most preferably ranges from 1.0to 4.0.

The term “electrolytic water,” when used herein, means water produced bythe process of electrolysis, and is preferably characterized by an oxidereduction potential (ORP) and/or pH that reflects its acid or alkalinenature.

The term “nanoclustered water,” when used herein, refers to water havinga reduced cluster size, typically induced by electrolysis. The size ofthe cluster can be measured by its NMR half line width, and in preferredembodiments the water has a NMR half line width using ¹⁷O of less thanabout 60, 56, or 52 Hz, preferably greater than about 42 or 45 Hz.

As used herein, “therapeutically effective amount” refers to an amountsufficient to elicit the desired biological response. Thetherapeutically effective amount or dose can depend on the age, sex andweight of the patient, and the current medical condition of the patient.The skilled artisan will be able to determine appropriate dosagesdepending on these and other factors in addition to the presentdisclosure.

The terms “treating” and “treatment,” when used herein, refer to themedical management of a patient with the intent to cure, ameliorate,stabilize, or prevent a disease, pathological condition, or disorder.This term includes active treatment, that is, treatment directedspecifically toward the improvement of a disease, pathologicalcondition, or disorder, and also includes causal treatment, that is,treatment directed toward removal of the cause of the associateddisease, pathological condition, or disorder. In addition, this termincludes palliative treatment, that is, treatment designed for therelief of symptoms rather than the curing of the disease, pathologicalcondition, or disorder; preventative treatment, that is, treatmentdirected to minimizing or partially or completely inhibiting thedevelopment of the associated disease, pathological condition, ordisorder; and supportive treatment, that is, treatment employed tosupplement another specific therapy directed toward the improvement ofthe associated disease, pathological condition, or disorder.

When the context allows, the term “significantly” can be interpreted tomean a level of statistical significance, in addition to“substantially.” The level of statistical significance can be, forexample, of at least p<0.05, of at least p<0.01, of at least p<0.005, orof at least p<0.001. When a measurable result or effect is expressed oridentified herein, it will be understood that the result or effect canbe evaluated based upon its statistical significance relative to abaseline.

The term “outer eye disorder,” as used herein, refers to any disorder ofthe cornea, iris or lens, that is associated with irregular growth orstructuring of protein or cellular components. Examples of outer eyedisorders include keratitis, neovascularization, epithelium deficiencyand cataracts. The keratitis can be superficial, ulcerative (i.e.corneal ulcer), hypopyon (i.e. hypopyon ulcer), mycotic (caused byfungus), or deep (perforating through all layers of cornea).Furthermore, the keratitis may result from bacteria, vitamin Adeficiencies, viruses, trauma (usually following insertion of an objectinto the eye), abrasion, surgery, fungi, or parasites. Theneovascularization can be localized, deep, or resulting in buildup oftissue (pannus). Furthermore, the neovascularization can result from, orbe associated with a lack of oxygen to the eye, trauma, abrasion,surgery, age related macular degeneration, inflammation and myopia.

The term “cataract,” as used herein, refers to a variety of conditionsthat create a cloudy or calcified lens that obstructs vision. Thecataract can be an infantile, juvenile, or presenile cataract.Alternatively, the cataract can be an age-related or senile cataract.Furthermore, the cataract can be either a traumatic cataract or acongenital cataract. The cataract can be located in a variety of regionswithin the lens. For example, the cataract can be an anteriorsubcapsular polar cataract (within the front, center lens surface), aposterior subcapsular polar cataract (within the rear, center lenssurface), a cortical cataract (radiating from the center to the edge ofthe lens), a nuclear cataract (in the center of the lens), orcombinations thereof. The cataract can also be associated with otherdisorders, such as diabetic cataract and toxic cataract. Furthermore,the cataract can be at a variety of stages such as immature cataract(partially opaque lens), mature cataract (completely opaque lens), orhypermature cataract (liquefied cortical matter, also known as aMorgagnian cataract).

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other additives, components, integers or steps.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” or like terms include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aningredient” includes mixtures of two or more ingredients, and the like.The word “or” or like terms as used herein means any one member of aparticular list and also includes any combination of members of thatlist.

Discussion

As discussed above, it has now been discovered that acidic electrolyzedwater can be used to effectively treat patients suffering from outer eyedisorders. This discovery is based on the results of experimentationdescribed in the “Examples” below, which revealed that a composition ofacidic nanoclustered water, unlike saline, affects the healing of theeye in a way that can be very useful in fostering correct protein andcellular structure and organization, and reducing opacity in the corneaand lens. It also has been discovered that acidic electrolyzed water ofa particular chemical composition can remain stable for significantlylonger period of time than previous electrolyzed oxidizing waters.

Therefore, in one embodiment, the invention provides a method oftreating an outer eye disorder selected from cataract, keratitis,corneal neovascularization and epithelium deficiency in an animalpatient in need thereof, comprising topically administering to an eye ofsaid patient a composition comprising a therapeutically effective amountof a high ORP acidic water. The composition is preferably in the form ofan eye drop.

Importantly, the method can decrease opacity of the lens, reduce loss ofvision, increase visual acuity, increase contrast sensitivity, andreduce halos. Therefore, in another embodiment, the invention provides amethod for improving opacity, reducing loss of vision, increasing visualacuity, increasing contrast sensitivity, or reducing halos in a lens ofan animal patient in need thereof, comprising administering to an eye ofsaid patient a composition comprising a therapeutically effective amountof a high ORP acidic water.

The step of administering the composition can be performed between 1 and20 times per day using any method known in the art, but is preferablyundertaken more than once during a 24 hour period. The administrationcan be carried out for a period of time including one week, 10 days, twoweeks, three weeks, one month, or continually as a maintenance therapy.In a particular embodiment, the administration step comprises droppingthe composition directly into the eye. In another embodiment, theadministration step comprises applying the composition to the eye withgauze. In yet another embodiment, the administering step comprisesapplying the composition to the eye with an eye washer.

The method can further comprise, before the administering step,diagnosing said eye as having a cataract or other outer eye disorder.

The step of administering the composition can be performed between 1 and4 times per day using any method known in the art. The administrationcan be carried out for a period of time including one week, 10 days, twoweeks, three weeks, one month, or continually as a maintenance therapy.In a particular embodiment, the administration step comprises droppingthe composition directly into the eye. In another embodiment, theadministration step comprises applying the composition to the eye withgauze. In yet another embodiment, the administering step comprisesapplying the composition to the eye with an eye washer.

The method can further comprise, before the administering step,performing surgery on said eye.

The Acid Waters

Acid water can be obtained with a water electrolysis method as describedbelow. The electrolytic acid waters can differ from similar productssubstantially in their stability, which is at least partly due to thehigher performance of the nano-coated electrodes and the electrolysisprocess. In conventional processes, even when the water is subjected toa filtration step before electrolysis, the electrodes tend to break upon their surface during the process, releasing large amounts of heavymetals (particularly of the metal or metals of which the cathode andanode are made). However, these acid waters can be free from heavymetals because said metals, if present, are present in a quantity whichis below the limits that can be detected with ordinary analyticalmethods. For example, the water according to the invention can have acadmium concentration of less than 5 μg/l, less than 10 μg/l ofchromium, less than 5 μg/l of lead, and less than 20 μg/l of nickel.Suitable test methods for these heavy metals are described in Table 1below:

TABLE 1 Heavy Metal Testing Methods TEST TESTING METHOD Cadmium APAT CNRIRSA 3120/2003 Total Chromium APAT CNR IRSA 3150/2003 Lead APAT CNR IRSA3230/2003 Nickel APAT CNR IRSA 3220/2003 Fixed Residue at 180° C. APATCNR IRSA 2090A/2003

Although one does not intend to be bound to any particular theory, it isbelieved that the absence of heavy metals is one of the main reasons forthe unusual and advantageous stability over time of the electrolyticacid water. The expression “stability over time” is used to mean thatthe acid water, if kept sheltered from the light, air and heat, keepsits chemical and physical properties, particularly its pH, ORP and/orNMR half line width, substantially unchanged for greater than 60 or 90days, preferably greater than 180 days, even more preferably greaterthan 365 days, up to two, three or even five years. By substantiallyunchanged, it is meant that the property under evaluation does not varyby more than 50, 30, 15, 10, 5, or even 3% during the applicable timeframe.

Although the stability time depends on the steps taken to preserve thesolution, it must be noted that for equal storage conditions, an acidicwater obtained by using an electrolytic device as defined above hasshown a distinctly higher stability than known similar products, whichin the best cases have shown a shelf life of only 60-90 days. Therefore,these products must be obtained and used over a short period or evensimultaneously with their production. Therefore, the electrolytic acidicwater according to the invention can be useful also for applications inlocations (Third World countries) and situations (scarcity of water toprovide electrolysis) in which favorable conditions for its productionare not available.

The ORP of the electrolytic acid water preferably ranges from +600 to+1350 mV, more preferably from +800, +900, 1000 or +1100 mV to +1300,1250 or +1200 mV, most preferably from +1100 to +1250 mV. The pH of theacid water preferably ranges from 0.5 or 1.0 to 6.5, 6.0, 5.0, 4.0, or3.0, and most preferably ranges from 1.0 to 4.0.

Nuclear magnetic resonance ¹⁷O NMR measures, particularly when evaluatedat the half way point of the water peak, are useful to measure thequality of acid waters of the current invention, because they reflectintrinsic properties of the water structure such as the median molecularcluster size of H₂O molecules, and the distribution of molecular clustersizes, in addition to contaminants such as ionic species within thewater. The expression “molecular cluster” designates the number ofmolecules of water which are coordinated in an ordered structure

In most preferred embodiments, the ¹⁷O NMR half line width for the acidwater is equal to or greater than 42, 45, 46, or 47, and less than 100,75, 60, 56, 53, 51, 50 or 49 Hz, wherein the range can be selected fromany of the foregoing endpoints. Thus, for example, in preferredembodiments, the acid water of the present invention has an NMR halfline width ranging from 45 to less than 51 Hz, or 45 to less than 50 Hz,or 46 to less than 50 Hz.

The acid water may also be characterized by the presence and quantity ofchlorine species in the water. One of the following assays or anycombination of the following assays may be used to characterize thewater. According to the free chlorine assay (spectrophotometric method),or the total chlorine assay (spectrophotometric method), the water maybe defined as containing less than 85, 70, 60, 55, 52 or even 50 mg/l ofchlorine species, optionally limited by a lower bound of 20, 30 or 40mg/l. According to the total chlorine assay (iodometric method), thewater may be defined as containing less than 80, 70, 65, or even 62 mg/lof chlorine species, optionally limited by a lower bound of 20, 30 or 40mg/l. According to the UNI 24012 (Mercurimetric method) chloride assay,the water may contain greater than 50, 100, 130, 150 or even 170 mg/l ofchloride, and/or less than 250 or 200 mg/l. Chlorites (as ClO²⁻), whenmeasured by EPA 300.1 (1997) (detection limit 100 ug/l), are preferablynon-detectable. Chlorates (ClO³⁻), when measured by EPA 300.1 (1997)(detection limit 0.1 mg/l), are preferably present in an amount lessthan 10, 5, 2, or even 1 mg/l.

Although in certain embodiments the acid water may contain oxidizingchlorine species in amounts of up to 60 or even 100 mg/l, in a preferredembodiment the acid water according to the invention is essentially freeof oxidizing chlorine species, or other anionic residues of salts thatare generated during the electrolytic process, i.e. less than 10 or even5 mg/l, and preferably undetectable.

In a particularly desirable embodiment, the water can be characterizedby conductivity, the presence of dissolved chlorine gas (Cl₂),hypochlorous acid (HOCl) and chloride ions (Cl⁻), and by the presence ofnegligible quantities of hypochlorite ion (OCl⁻). In water, the relativeamount of chlorine and hypochlorous acid is strongly affected by theamount of chlorides. Specifically, an increase in chlorides results inan increase in the amount of chlorine gas with respect to hypochlorousacid as according to the following equilibrium:

Cl⁻+H⁺+HOCl

Cl₂+H₂O

Because of the relationship between the amount of chlorides and theamounts of chlorine gas and hypochlorous acid, the amount of chloride inthe water is preferably very low (less than 200 ppm) to ensure that thefree chlorine in the water is almost exclusively in the form ofhypochlorous acid.

The relationship between the four species Cl₂, HOCl, Cl⁻, and OCl⁻ canbe understood using the disassociation equilibria of gaseous chlorine inwater as described below, in which Cl₂, HOCl, and OCl⁻ are the threepossible forms of free total chlorine:

Cl₂+H₂O=Cl⁻+H⁺+HOCl K_(a1)≈3×10⁻⁴

HOCl=H⁺+OCl⁻K_(a2)≈2.9×10⁻⁸

As can be seen in the above equations, chlorine generation occurs in thepresence of an excess of Cl⁻. Furthermore, the amount of the three formsof free total chlorine as a function of pH and Cl⁻ can be determinedalgebraically by using the above described equilibria as follows:

αCl₂=[H⁺]²[Cl⁻]/([H⁺]²[Cl⁻]+[H⁺]K_(a1)+K_(a1)K_(a2))

αHClO=[H⁺]K_(a1)/([H⁺]²[Cl⁻]+[H⁺]K_(a1)+K_(a1)K_(a2))

αClO⁻=K_(a1)K_(a2)/([H⁺]²[Cl⁻]+[H⁺]K_(a1)+K_(a1)K_(a2))

These equations can be used to simulate the chlorine concentration atdifferent pH values. For example, FIG. 3 is a graphical simulation ofthe concentration of various chlorine species as a function of pH. Ascan be seen in FIG. 3, at a typical pH for the water of 2.8-3.0(indicated by the green dash-and-dotted line), free chlorine ispredominantly present as Cl₂ and HClO, and the relative amount of thetwo species is strongly affected by the amount of chlorides, andincrease of which results in an increase of the amount of chlorine gaswith respect to HClO.

It is generally recognized that diluted hypochlorous acid solutions areunstable due to decomposition. This decomposition can occur according toa first pathway:

2HOCl→2HCl+O₂

2HCl+2HOCl→2Cl₂+2H₂O

Or as according to a second pathway, in which chlorous acid (HClO₂) isan intermediate in the formation of chloric acid (HClO₃):

2HOCl→[HClO₂]+HCl

2HOCl+[HClO₂]→HClO₃+HCl

HClO₃+HCl+HOCl→HClO₃+Cl₂+H₂O

Kinetic studies have indicated that both decomposition pathways are pHdependant and increase with concentration, temperature, and exposure tolight. Furthermore, the first process can be accelerated by catalysts,and the second process can be accelerated in the presence of otherelectrolytes, notably chloride ions. Due to decomposition, hypochloritesolutions can be more stable than hypochlorous acid solutions. For thisreason, commercial solutions often have neutral or alkaline pH, whichcauses the free chlorine to exist as hypochlorite and not hypochlorousacid.

Although one does not intend to be bound to any particular theory, it isbelieved that the low chloride ion content is one of the main reasonsfor the unusual and advantageous stability over time of the electrolyticacid water, both to evaporation and self decomposition. Preferably, theamount of chlorides both at the beginning and at the end of theelectrolytic process is low (200 ppm or lower), so that the watercomprises chlorine in the form of HClO. For example, in a particularembodiment, the water can comprise ˜50 ppm of free chlorine, and ˜200ppm of chloride ions. At pH 2.80, this corresponds to 99.3% HClO, and0.7% dissolved gaseous chlorine.

The conductivity of the water preferably ranges from 900 to 1800 uS/cm,and more preferably ranges from 1000, 1100, 1200, or 1300 to 1400, 1500,1600, or 1700 uS/cm. The free chlorine content of the water preferablyranges from 20 to 80 ppm, more preferably ranges from 30 or 40 to 60 or70 ppm, and most preferably is about 50 ppm. The chloride ion content ofthe water preferably ranges from 150 to 250 ppm, more preferably rangesfrom 160, 170, 180 or 190 to 210, 220, 230, or 240 ppm, and mostpreferably is about 200 ppm. The chlorite content of the waterpreferably ranges from 50 to 150 ppb, more preferably ranges from 60,70, 80 or 90 to 110, 120, 130, or 140 ppb, and most preferably is about100 ppb. The chlorate content of the water preferably ranges from 0.5 to1.5 ppm, more preferably ranges from 0.6, 0.7, 0.8, or 0.9 to 1.1, 1.2,1.3, or 1.4 ppb, and most preferably is about 1 ppm.

Due to its chemical composition and acidity, the free chlorine in thewater can be present in the form of hypochlorous acid (HOCl) andchlorine gas (Cl₂). The relative amount of HOCl and Cl₂ in the waterpreferably ranges from 99.9% HOCl and 0.1% Cl₂ to 95% HOCl to 5% Cl₂,more preferably ranges from 99.5% HOCl and 0.5% Cl₂ to 98.5% HOCl to1.5% Cl₂, and most preferably is about 99.3% HOCl and 0.7% Cl₂.

Because the free chlorine in the water is present in the form of HOCland Cl₂ in the ranges described above, the water can be highly stableagainst both evaporation and self decomposition. In an exposed,non-agitated state at a temperature of 25° C., the water preferablymaintains a level of chlorine for a time of 2, 3, 4, 5, 6, 7, 8, 9, or10 days. In an exposed, agitated state at a temperature of 25° C., thewater preferably maintains a level of chlorine for a time of 8, 12, 16,20, or 24 hours. In a closed state at a temperature of 25° C., the waterpreferably maintains about 90% of the free chlorine after a time of 3months, and about 85% of the free chlorine after a time of 12 months. Ina closed state at a temperature of 30° C., the water preferablymaintains about 90% of the free chlorine after a time of 3 months, andabout 80% of the free chlorine after a time of 12 months. In a closedstate at a temperature of 40° C., the water preferably maintains about90% of the free chlorine after a time of 3 months, and about 85% of thefree chlorine after a time of 12 months.

Making Electrolytic Acid Water

The electrolytic acid water can be prepared, for example, by using themethods and electrolysis devices described in PCT Publications WO2008/131936 and WO 2007/048772. The contents of said applications arehereby incorporated by reference as if fully set forth herein.

Referring now to FIG. 1, the electrolysis device can comprise anelectrolysis chamber 3 divided into two portions by a membrane 4, and apair of electrodes 1 and 2 within said chamber.

Preferably, both electrodes of the device are nano-coated electrodes asdefined below. However, the advantages in terms of low cost andefficiency of the electrolysis process, as well as the advantages interms of water stability over time, can be obtained also if only one ofthe two electrodes is nano-coated as defined above.

Preferably, the device according to the invention also comprises amembrane 4 adapted to divide the at least one chamber into twohalf-chambers, wherein the half-chamber that contains the anode istermed an anode half-chamber, and the half-chamber that contains thecathode is termed a cathode half-chamber. The membrane is advantageouslyan ultrafiltration membrane which can occupy the chamber partially ortotally.

The membrane 4 can be of the type used in conventional electrolyticcells, but is preferably based on size exclusion technology at thenano-scale. Preferably, the membrane is made of ceramic material withopen porosity, coated with metallic nano-particles, preferablynano-particles of oxides of zirconium, yttrium, aluminum or mixturesthereof. The metallic nano-particles used to make the coating arepreferably in powder form. As regards the size distribution within thepowder, preferably an amount at least equal to 70%, 75%, or 80% byweight of the particles that are present in the powder, more preferablyat least equal to 85%, have a particle diameter ranging from 30 to 100nm, 40 to 70 nm, or 50 to 60 nm.

By resorting to nanometer particles to manufacture the membrane 4, theaverage pore size of the final membrane has been found to be extremelyconstant over time and adaptable according to the requirements of howthe water is to be processed. Preferably, the average pore size is fromabout 120 to about 180 nm (mean or median). Size constancy over time andconstancy of the pore dimensions themselves are two aspects whichdifferentiate the ceramic membrane described here from the textilemembranes conventionally used in equivalent devices (which are insteadsubject to rapid deterioration over time). It is preferred that at least50%, 70%, 90%, 95%, 98% or 99% of the pores have a diameter between 120and 180 nm. These aspects have shown a positive effect on the stabilityof the water obtained after electrolysis, where this effect combineswith, and augments, the stabilizing effect produced by the use of anelectrode as defined above.

Importantly, the nano-sized dimensional features of the membrane andelectrodes enhance the amount of active surface per unit of geometricsurface, which creates a high apparent current density (i.e. the currentintensity per unit of geometric surface). As a result, a high currentintensity (ampere) and electric potential (voltage) can be provided tothe solution, which can impart unique chemical and biologicalcharacteristics to the water. Preferably, the water is produced byapplying a current intensity in the range of about 100 to about 39ampere (24 to 18 volt) to a diluted sodium chloride solution indeionized water. By applying the high current intensity, a chemicalcomposition of a low chloride ion content, low chlorite and chloratecontent, and high hypochlorous acid content can be achieved.

The amount of current applied to the water preferably ranges from 30 to120 ampere, more preferably ranges from 40, 50 or 60 ampere to 90, 100,or 110 ampere, and most preferably is about 80 ampere. The amount ofvoltage applied to the water preferably ranges from 15 to 35 volt, morepreferably ranges from 16, 17, or 18 volt to 22, 23, or 24 volt, andmost preferably is about 20 volt.

In a preferred electrolysis device, each half-chamber is connected tothe outside of the device through:

-   -   openings 7 and 8 arranged in the upper part of the half-chamber        from which the water to be subjected to electrolysis is        inserted, and    -   additional openings 5 and 6 arranged in the lower part of the        half-chamber which can act as a discharge for the resulting acid        and alkaline fractions (referenced as “acid water” and        “alkalescent water” in FIG. 1). The second opening on the lower        part of each half chamber is provided with closure means (not        shown) which is adapted to prevent the water that has not yet        separated from leaving the half-chamber and are adapted to be        opened at the end of the electrolytic process.

With specific reference to FIG. 1, the operating mechanism of a deviceas described above provided with all the essential and optional elementsthat have been listed, therefore entails treating water by introducingit from above, by means of the water input ducts, into the twohalf-chambers of the main chamber. Here, the water, under the action ofthe cathode and of the anode previously connected to the negative andpositive poles of an electric voltage source, is split into positive andnegative ions, which, as is known, are attracted by the respectiveopposite poles. In passing from one half-chamber to the other, thenano-porous membrane acts as a filter for said ions and for any chargedparticles, allowing only the particles of sufficiently small size topass.

The water input to the unit can be characterized by its conductivity,preferably measured in μS/cm. Thus, for example, the water can bedescribed by the consistency of conductivity in the water input. Forexample, the conductivity should vary by no more than 50, 20, 10, 5 oreven 2 μS/cm, or 100, 50, 20 or 10%. The water may also be described bythe conductivity of the water itself. The conductivity can range from0.5, 1.0 or 1.5 μS/cm to 50, 25, 10, 5 or even 3 μS/cm, based on anyselection of endpoints. The conductivity preferably ranges from 0.5 to10 or 0.5 to 3 μS/cm, and the most preferred conductivity is about 2μS/cm. It has been discovered that by controlling the consistency of theconductivity, and by lowering the conductivity to the preferred values,one is able to obtain much more consistent quality electrolyzed water,with a consequent reduction in NMR half line width. Suitable types ofwater for input into the unit include reverse osmosis water, deionizedwater, and distilled water. A preferred type of water due to itsconstant conductivity is osmotic water prepared by reverse osmosis.

The water preferably contains sodium chloride, or some other alkalimetal salt, to facilitate the electrolysis. The sodium chloride ispreferably pharmaceutical grade. The quantity of sodium chloridecontained in the water is such that the water obtains a specific levelof conductivity. The conductivity of the input solution preferablyranges from 50 μS/cm to 100 μS/cm, more preferably ranges from 150 μS/cmto 200 μS/cm, and most preferably is about 200 μS/cm.

Also of importance, the filter prevents the transmission of heavy metalsfrom one chamber to the other. Thus, by introducing the water into theacidic or alkaline chamber, one is able to produce alkaline or acidwater having practically no contamination by metallic radicals (or atleast beyond the limits of detection).

A method of using such a unit for making electrolytic acid water havinga NMR half line width using ¹⁷O-NMR of from about 45 to less than 51 Hzcomprises:

(a) providing an electrolysis unit comprising: (i) a cathode chamber, ananode chamber, and a filter separating said chambers (preferablycharacterized by a porosity that allows ionized fractions ofnano-clustered H₂O to pass, such as when the porosity is predominantlycharacterized by pores of from about 120 to about 180 nm in diameter(preferably having a mean diameter between 120 and 180 nm)); and (ii) acathode situated in said cathode chamber and an anode situated withinsaid anode chamber, wherein at least one of said anode and cathode iscoated by a residue of particles in which greater than 70% by weight ofsaid particles have a diameter of from 40 to 100 nm;

(b) introducing a solution of water and an alkali metal into one or bothof said chambers; and

(c) applying an electric potential to said anode and said cathode, for atime and to an extent sufficient to produce electrolyzed acidic waterhaving a NMR half line width using ¹⁷O of from about 45 to less than 51Hz.

Electrode Construction

Referring again to FIG. 1, the electrolysis device includes electrodes 1and 2 that comprise a surface coating which comprises nano-particles ofone or more metals. Preferably, the electrodes comprise a core which ismade of a metallic material, a nonmetallic material or combinationsthereof.

If the core is made of metallic material, it can be made for example ofan alloy of titanium and platinum or an alloy of steel and graphite. Ifthe core is made of a nonmetallic material, it can be made for exampleof graphite. The core may also comprise different layers, such as forexample a core made of graphite which is coated with an outer layer ofmetal, for example titanium. The term “metal” references both a metaland chemical compounds which comprise said metal, such as its oxides. Apreferred core is made of TiO₂.

The electrode can be characterized with respect to known electrodessubstantially due to the presence of a nanometer covering (hereinafteralso referenced as coating) which is extremely smooth, i.e., a layer forcovering the core which includes metallic nano-particles.

The metals of which the nano-particles of the coating are made areselected preferably among one or more of titanium, iridium, yttrium,ruthenium, zinc, zirconium platinum, selenium, tantalum and compoundsthereof. Preferred metal compounds are oxides of the mentioned metals. Apreferred coating comprises ZrO₂, ZnO, Ru₂O₃, IrO₂ and Y₂O₃, or TiO₂,Pt/ZrO₂, SnO₂, Ta₂O₅, and IrO₂. Preferably, the various metals are usedin powder form.

The coating can also comprise a nonmetallic carrier material, forexample particles of one or more polymers. The polymer can be synthetic(such as for example plastics, acrylic polymers, et cetera) or partlysynthetic (such as for example modified celluloses, modified starches,et cetera). The metallic nano-particles comprised within the coating arepreferably used in powder form. As regards the size distribution withinthe powder, preferably an amount at least equal to 70%, 75%, or 80% byweight of the particles that are present in the powder, more preferablyat least equal to 85%, has a particle diameter ranging from 40 to 100nm, 50 to 90 nm, or 60 to 80 nm.

The electrode coating can be provided by means of nanotechnologytechniques which are known to a person skilled in the art and areadapted to produce a smooth surface, for example by sintering the powderor the mixtures of metallic nano-powders.

The individual metals in powder form can be applied to the electrode soas to produce the coating: 1) as a preformed mixture, and/or 2) in theform of discrete layers which are applied sequentially and mutuallysuperimposed and wherein each layer consists of a single metal, and/or3) in the form of discrete layers which are applied sequentially andmutually superimposed and in which each layer consists of two or moremetals but not simultaneously of all the metals that are present in thecoating.

Preferably, the method comprises the step (A) of preparing the coatingof the electrode by sintering powders of nano-particles of one or moremetals as defined above directly on the core of the electrode.Preferably, step (A) comprises the following steps to be performed inthe order in which they are listed here:

(A1) preparing one or more powders of metallic nano-particles as definedabove,

(A2) dissolving the one or more powders of nano-particles in a suitablesolvent and in at least such a quantity as to be able to dissolve allthe powder to be applied, obtaining one or more solutions, and

(A3) sintering the one or more solutions obtained in the preceding stepon a metal plate, preferably passivated on its surface, which will formthe core of the electrode.

Preferably:

-   -   the one or more powders of metallic nano-particles of step (A1)        is a combination of powders of ZrO₂, ZnO, Ru₂O₃, IrO₂ and Y₂O₃,        or TiO₂, Pt/ZrO₂, SnO₂, Ta₂O₅, and IrO₂, advantageously obtained        by hydrothermal chemical processing, at least 70%, 75%, or 80%        and more preferably at least 85% by weight of the particles in        the powder have a diameter ranging from 60 to 80 nm;    -   the solvent of step (A2) in which each powder is dissolved is        preferably a 30% solution by weight of hydrochloric acid in        water, in at least such an amount as to be able to dissolve all        the powder to be applied,    -   step (A3) consists in sintering the aqueous solutions of        hydrochloric acid obtained from step A(2) on both faces of a        TiO₂ plate which is passivated on its surface and has a        thickness ranging from 0.15 to 0.35 mm, wherein sintering may        occur according to the steps listed below in Table 2:

TABLE 2 Sintering Steps Sintering Dosage per unit Sintering temperatureStep Solution surface time (min) (° C.) 1 IrO₂  0.2 g/m² 45 450 2 Ru₂O₃ 0.2 g/m² 45 450 3 ZnO + Y₂O₃ 0.15 g/m 60 550 (Y at 2 mol) 4 IrO₂ 0.25g/m² 45 450 5 Ru₂O₃ 0.25 g/m² 60 550 6 ZrO₂ + Y₂O₃  0.1 g/m² 60 550 (Yat 3 mol) 7 Ru₂O₃ 0.15 g/m² 60 550 8 IrO₂ 0.15 g/m² 60 550 9 IrO₂ +Ru₂O₃ 0.15 g/m² + 0.15 g/m² 60 600 10 ZrO₂ + Y₂O₃  0.1 g/m 60 600 (Y at3 mol) 11 IrO₂ + Ru₂O₃ 0.15 g/m² + 0.15 g/m² 60 600

Resorting to multiple sintering steps has been found to be particularlyuseful in order to eliminate any roughness from the surface of theelectrode and obtain an extremely hard and smooth surface. An electrodeas defined above, used as part of a device for providing theelectrolysis of water, produces the following advantages:

-   -   more efficient electrolysis, in that there is a lower        consumption of salts such as NaCl, used conventionally to        accelerate the electrolysis of low-conductivity fluids such as        water; and    -   if both electrodes are electrodes according to the invention,        the possibility to provide a continuous change of polarity of        the electrodes (“polarity swapping”). The sudden change of        polarity allows the charged particles that are present in the        fluid subjected to electrolysis to circulate in both directions        instead of just in one (forced by the charge of the particles        and by the unchangeable sign of the electrodes), thus avoiding        the forming of deposit-producing masses at the level of the        electrodes and thus keeping their surface clean and their        efficiency at the maximum level. Moreover, if a semipermeable        membrane is provided within the electrolytic cell and divides        the two anode and cathode half-chambers, the change of polarity        avoids the clogging of the pores of said membrane, extending the        life of the device;    -   the presence of a nanometer coating determines an accumulation        of charge by the upper electrode to more than 100% with respect        to conventional electrodes. This allows to provide a        qualitatively and quantitatively different electrolysis at        significantly higher potentials, with the effect of, for        example, reducing the size of molecular clusters;    -   the obtainment of a very high consistency, smoothness and        surface density, aspects which avoid the solubilization of the        electrode itself or the forming of sediments on its surface,        which would then occur in the acid and alkaline water fractions.        The same aspects are also the basis for the substantially nil        release of heavy metals and other compounds which constitute the        surface and core of the electrode within the acid and alkaline        water fractions. As will be mentioned also hereafter, the        absence of heavy metals in the water leads to an amazing        stability thereof over time, with preservation of        characteristics such as ORP, pH and molecular cluster size. This        stability is unknown to known equivalent products. The same        aspects are also the basis for the minimal maintenance required        by the electrode, which can be changed with a significantly        lower frequency than known electrodes, reducing costs and        increasing ease of production;    -   the possibility to obtain quantum effects (known in the        literature also by the term “nano-effects”) by means of the        nanometer dimensions of the coating particles. Briefly, when        nanometer dimensions are reached, the optical, magnetic and        electrical properties of matter change radically. By reducing        the dimensions until the typical nanometer dimensions of        so-called clusters are reached, due to the small number of atoms        that are present in said cluster and to its reduced volume, a        discretization of the energy levels (quantization) becomes        apparent in the electron structure and depends on the size of        the cluster, this phenomenon is known as “quantum size effect”        and entirely new characteristics, which contrast with the ones        that are typical of the material at ordinary dimensions, depend        from it. In the present case, the best performance has been        obtained with powders which have a size distribution centered in        an interval ranging from 60 to 80 nm as indicated above. As a        whole, the effects described above produce the simultaneous        presence of three factors: stability of the resulting water,        ease of its production (for example thanks to the lower        maintenance costs and to the greater durability of the device as        a whole) and an increase in its quality (especially in terms of        purity and constancy of properties over time). In particular,        the increase in the quality of the water can be measured both in        terms of uniformity of the dimensions of the molecular clusters        (higher percentage of micromolecules with respect to the number        of macromolecular clusters) and in terms of increased stability        over time of the properties given to the water by the        electrolysis itself (above all pH, ORP and cluster size). The        stability increase presumably achieves the preservation over        time of the structural surface characteristics of the electrodes        coated with a nano-coating as described here.

EXAMPLES Example 1 Objective

Determine the efficacy of acidic nanoclustered water (ANW) againstseveral strains of bacteria, viruses and fungi.

Bacterial Activity

Bacterial activity was assessed with the method of UNI (ItalianOrganization for Standardization) EN 1040 (quantitative suspension testfor the evaluation of basic bactericidal activity of chemicaldisinfectants and antiseptics). According to this method, a substance isclassified as bactericidal for a specific microorganism if it reducesthe bacterial count by at least 5-log₁₀ following 5 minutes of contactat 20° C. ANW solutions at three different concentrations (80%, 50%, and25%) were tested against two strains of bacteria known to cause eyeinfections, Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa(ATCC 15442). Table 3 below shows the antibacterial effect of the threedifferent concentrations of ANW, with viability reduction valuesexpressed as the log₁₀ reduction.

TABLE 3 Antibacterial Effect of ANW Viability Reduction Species Solution80% 50% 25% Staphylococcus aureus ANW >5.41 >5.41 5.35 (ATCC 6538)Pseudomonas aeruginosa ANW >5.48 <4.10 <4.10 (ATCC 15442)

As shown in Table 3, ANW can be classified to be a bactericidal againstboth strains at a concentration of 80%.

Bacterial activity was also assessed against the same two strains ofbacteria in the presence of 5% of human blood in the medium as organicsoil interference. Viability reduction was assessed after 10, 30, 60 and120 minutes of exposure to pure ANW at 31° C. Table 4 below shows theantibacterial effect of the pure ANW at each time point, with viabilityreduction values expressed as the log₁₀ reduction.

TABLE 4 Antibacterial Effect of ANW in Presence of 5% Human BloodViability Reduction Species Solution 10 min 30 min 60 min 120 minStaphylococcus aureus ANW >5.6 >5.6 >5.6 >5.6 (ATCC 6538) Pseudomonasaeruginosa ANW >5.6 >5.6 >5.6 >5.6 (ATCC 15442)

As shown in Table 4, pure ANW was demonstrated to have a bactericidaleffect against both strains at the lowest tested time point of 10minutes.

Bacterial activity was also assessed against Propionibacterium acnesbacteria in the presence of 1% fetal bovine serum in the medium asorganic soil interference. Viability reduction was assessed after 1, 5,15 and 30 minutes of exposure to pure ANW at 31° C. Table 5 below showsthe antibacterial effect of the pure ANW at each time point, withviability reduction values expressed as the log₁₀ reduction.

TABLE 5 Antibacterial Effect of ANW in Presence of 1% Fetal Bovine SerumViability Reduction Species Solution 1 min 5 min 15 min 30 minPropionibacterium acnes ANW >6.9 >6.9 5.3 >6.9 (ATCC 11827)

As shown in Table 5, pure ANW was demonstrated to have a bactericidaleffect at the lowest tested time point of 1 minute.

Fungal Activity

Fungal activity was assessed with the method of UNI EN 1275(quantitative suspension test for the evaluation of basic fungicidalactivity of chemical disinfectants and antiseptics). According to thismethod, a substance is classified as fungicidal for a specificmicroorganism if it reduces the fungi count by at least 4-log₁₀following 15 minutes of contact at 20° C. ANW solutions at threedifferent concentrations (80%, 50%, and 25%) were tested against twostrains of fungus known to cause infections, Candida albicans (ATCC10231) and Aspergillus niger (ATCC 16404). Table 6 below shows theantifungal effect of the three different concentrations of ANW, withviability reduction values expressed as the log₁₀ reduction.

TABLE 6 Antifungal Effect of ANW Viability Reduction Species Solution80% 50% 25% Candida albicans (ATCC ANW >4.37 <3.09 <3.09 10231)Aspergillus niger (ATCC ANW <3.12 <3.12 <3.12 16404)

As shown in Table 6, ANW can be classified to be a bactericidal againstCandida albicans (ATCC 10231) at a concentration of 80%.

Viral Activity

Viral activity was assessed against Human Immunodeficiency Virus type 1(HIV-1), Herpes Simplex Virus type 1 (HSV-1), and Herpes Simplex Virustype 2 (HSV-2) in the presence of 5% fetal bovine serum in the medium asorganic soil interference. For HIV-1, viability reduction was assessedafter 10 minutes of exposure to pure ANW at 21.5° C. For HSV-1 andHSV-2, viability reduction was assessed after 5 minutes of exposure topure ANW at 35° C. Table 7 below shows the antiviral effect of the pureANW on each virus, with viability reduction values expressed as thelog₁₀ reduction.

TABLE 7 Antiviral Effect of ANW Viability Species Solution ExposureReduction HIV-1 (strain HTLV-III_(b)) ANW 10 min at 21.5° C. >4.5 HSV-1(ATCC VR733) ANW  5 min at 35° C. <5.5 HSV-2 (ATCC VR734) ANW  5 min at35° C. 4.25

As shown in Table 7, ANW can inactivate the tested viruses.

Example 2 Objective

Determine the efficacy of acidic nanoclustered water (ANW) at promotingcorneal healing and cataract healing.

Corneal Ulceration Healing

Corneal healing activity was assessed in an in vivo rabbit model.Corneal eye wounds were experimentally provoked in 8 rabbits. The leftand right eyes were then treated with ANW and saline, respectively, byapplying 100 μl of the solutions 4 times per day for 14 consecutivedays. On the 4^(th), 9^(th), and 14^(th) day after the surgery, imagesof each wound were taken under a slit lamp microscope and the area ofeach wound was calculated with the software Topcon IMAGENET 2000. Thewound area was then used to calculate the wound healing rate (WHR) usingthe following equation:

WHR=100(1−(wound area at timing point)/(initial wound area))

Table 8 below shows the Wound Healing Rate of ANW and saline treatedeyes at 4, 9, and 14 days.

TABLE 8 Corneal Ulceration Healing Effect of ANW and Saline WoundHealing Rate (WHR) Day 4 Day 9 Day 14 ANW 77.78 ± 9.06  83.32 ± 12.2387.20 ± 13.16 Saline 71.84 ± 19.38 63.45 ± 23.02 64.23 ± 28.28 T-value1.36 3.73 3.61 P-value >0.05 <0.05 <0.05

As shown in Table 8, ANW was significantly more effective than saline incorneal ulcer healing at the latter two of the three time points.

The wounds were also observed daily for the presence of wound closure.On day 14, it was observed that half of the corneal ulcers treated withANW were healed, while some corneas treated with saline were stillpresenting a large ulcer. Exemplary photographs (not shown) were takenof the corneal wounds in 3 of the rabbits on day 14.

The wounds were also observed daily for the presence of infections andinflammation. ANW was observed to reduce inflammation after injury.Furthermore, two of the eyes treated with saline were seriously infectedwith hypopyon, and the inflammation of these corneas was too intensiveto identify the pupil. Exemplary photographs and histological images(not shown) were obtained of the inflammation in 2 of the rabbits.

Histological evaluation also was used to observe regeneration of thecornea and scarring. ANW was observed to increase regeneration andreduce scarring as compared to saline. Furthermore, epitheliumdeficiency was observed in all of the eyes treated with saline, and noneof the eyes treated with ANW. Histological images (not shown) depictingscarring of cornea wounds were taken in 3 of the rabbits.

The wounds were also observed for the presence of angiogenesis.Neovascularization was observed in 35% of the corneas treated withsaline, and none of the eyes treated with ANW. Exemplary photographs(not shown) depicting angiogenesis were taken of 2 of the rabbits.

Cataract Healing

Cataract healing activity was assessed in an in vivo rat model. Cataractwas induced by intraperitoneal injection of d-galactose in 1 rat at adose of 10 g/kg per day (twice/day). The left and right eyes were thentreated with ANW and saline, respectively, by applying 1 drop of thesolutions 4 times per day for 30 consecutive days. On day 30, it wasobserved that the cataract treated with ANW was significantly healed ascompared to the cataract treated with saline. Photographs (not shown)depicting the cataracts were taken on day 30.

Example 3 Objective

Determine the safety of acidic nanoclustered water (ANW) in systemic andtopical applications.

In Vitro Studies

Citotoxicity was assessed with the method of ISO (InternationalOrganization for Standardization) 10993-5. According to this method, asubstance is classified based on its effect on a cell culture. 100 μl ofpure ANW was applied to a cell culture of murine fibroblasts L-929 andthe cells were evaluated after 24 hours of incubation at 37° C. Somemalformed cells were observed after the period of incubation. Based onthese results, ANW was defined as “slightly cytotoxic” (grade 1 of 4).

Mutagenicity was assessed with the method of OECD 471. According to thismethod, a substance is classified based on its ability to induce pointmutations in bacteria. Five mutant strains of Salmonella typhimurium (TA1535, TA 1537, TA 98, TA 100, and TA 102) were studied both in thepresence and in the absence of ANW. Based on the results of a reversemutation assay (Ames' test), the substance ANW was defined as nonmutagenic.

Systemic Toxicity Studies

Acute toxicity was assessed with the method of ISO 10993-11, 2006,Biological Evaluation of Medical Devices—Part 11: Tests for SystemicToxicity. According to this method, a substance is classified not toxicif animals injected with the substance do not show a significantlygreater biological reaction than animals treated with a control article.10 female Swiss albino mice were injected by intraperitoneal route witheither ANW or saline in the amount of 50 mL/kg. The animals wereobserved for clinical signs immediately after injection, and at 4, 24,48, and 72±2 hours after injection. ANW did not induce a significantlygreater biological reaction than the control, and was thereforeclassified as not toxic.

Skin Irritation Studies

Dermal irritation following acute exposures was assessed with the methodof ISO 10993-10. 0.5 mL of pure ANW was applied with a patch on theshaved skin of three male albino rabbits. The patch was held on the skinby means of a non-irritating adhesive plaster for 4 hours. The skinreaction was observed upon removal of the patch and 24, 48, and 72 hoursafter removal. No sign of either erythema or edema was observed. Basedon these results, ANW was determined to be non-irritating for skin,which a Skin Irritation Index of 0.00.

Dermal irritation following repeated exposure also was assessed with themethod of ISO 10993-10. Three male New Zealand rabbits were treated 5days a week for 4 weeks with two consecutive daily administrations of 5mL of pure ANW or saline as a control applied with a patch for one hour.The skin reaction was before and after each application throughout theentire 4 week period. No sign of either erythema or edema was observed.Furthermore, upon sacrifice, no signs of inflammation were detected inhistological images. Based on these results, ANW was determined to notexhibit any significant irritancy in the skin.

Skin Sensitization Studies

Delayed-type skin hypersensitivity was assessed with the method of ISO10993-10: Guinea-Pig Maximization test. The test used 15 albino femaleHartley guinea pigs (10 treated and 5 control). The injection phase (Day0) was carried out by administering three 0.1 mL intradermal injectionsto each animal: (a) complete Freund's adjuvant, (b) either pure ANW(test) or saline (control), (c) either ANW (test) or saline (control)mixed together with complete Freund's adjuvant. A skin massage with 1 mLSLS 10% was then performed on Day 6. The induction phase (Day 7) wascarried out by applying 1 mL of either the test or the control, left inplace for 48 hours with an occlusive patch. The challenge was carriedout on Day 21 through the application to each animal (both treated andcontrol) of dressings with of 1 mL of ANW on the right side and 1 mL ofsaline on the left side, left in place for 24 hours.

Assessments were carried out on the 23rd day (24 hours after patch andremoval) and on the 24th day (48 hours after patch and removal). Theintensity of erythema and/or edema was evaluated according to theMagnusson and Kligman scale from 0 to 3.

No abnormalities were observed in either the treated or the controlanimals. Based on these results, ANW was determined to not exhibitdelayed contact dermatitis potential.

Ocular Irritation Studies

Ocular irritation was assessed with the method of ISO 10993-10. In afirst experiment, three New Zealand white rabbits were treated byinstilling 0.1 mL of pure ANW into the left eye, leaving the right eyeuntreated as a control. The eyes were examined 1, 24, 48 and 72 hoursafter instillation through fluorescein staining and slit-lampobservation. No signs of irritation were observed in any of the eyes.Based on these results, ANW was determined to be a non-irritant for theocular tissue of New Zealand White rabbits.

In a second experiment, ocular irritation was again assessed with themethod of ISO 10993-10. Three New Zealand white rabbits were treated byinstilling 0.1 mL of pure ANW into the left eye as a test, andinstilling 0.1 mL of NaCl containing water (saline) into the right eyeas a control. The treatment was repeated for 30 consecutive days. Nosigns of irritation were observed in any of the test or control eyes atany of the observation points. Based on these results, the test articleANW was determined to be a non-irritant for the ocular tissue of NewZealand White rabbits.

Summary of Toxicology Studies

Table 9 below shows a synopsis of the safety studies reported above inExample 3.

TABLE 9 Synopsis of ANW Safety Studies Effect Method ResultsCytotoxicity In vitro mouse fibroblasts Slightly cytotoxic L-929 (grade1 of 4) Acute Skin Irritation Acute exposure in rabbits Non-irritantRepeated Skin Irritation Repeated exposure Non-irritant in rabbitsDelayed hypersensitivity Maximisation test in Non-sensitising guineapigs Primary ocular irritation Acute administration Non-irritant inrabbits Repeated ocular irritation Repeated administration Non-irritantin rabbits Genotoxicity Ames test Non-mutagenic Acute Systemic Toxicityi.p. dosing in mice Not toxic up to 50 mL/kg i.p.

Example 4 Objective

Determine the efficacy of acidic nanoclustered water (ANW) formodulating the activity of the immune system

In Vitro Study of PBMC Proliferation

The ability of ANW to inhibit the proliferation of peripheral bloodmononuclear cells (PBMC) was assessed in an in vitro cellular modelusing 12 batches of PCMC. In the experiment, 4 of the batches wereexposed to betamethasone (10 nM), a glucocorticoid steroid withanti-inflammatory and immunosuppressive properties, 4 of the batcheswere exposed to a 1:10 dilution of ANW, and the remaining 4 batches wereexposed to a 1:20 dilution of ANW. Table 10 below shows the inhibitioneffect that was measured for each of the 12 batches.

TABLE 10 Inhibition of PBMC Proliferation Batch 1 Batch 2 Batch 3 Batch4 Mean Betamethasone (10 nM) 87.7% 85.7% 79.4% 88.5% 85.3% AcidicNanoclustered 39.9% 29.0% 19.3% 16.6% 26.2% Water (1:10) AcidicNanoclustered 17.3% 22.0%  4.0%  7.0% 12.6% Water (1:20)

As shown in Table 10, ANW inhibited PBMC proliferation at bothdilutions. These dilutions have previously been shown to not besignificantly toxic on this cell type in vitro.

In Vitro Study of T-Cell Activation

The ability of ANW to modulate immunoregulatory cytokines was assessedin an in vitro cellular model using PBMC from 4 blood donors stimulatedwith purified protein derivatives from Mycobacterium tuberculosis (PPD),a prototypical Th1 antigen. In the experiment, batches of PBMC cellswere stimulated with PPD alone, PPD plus betamethasone (10 nM), and PPDplus Acidic Nanoclustered Water (1:10 dilution). T-Cell activation wasmeasured by testing levels of three cytokines: IL-10 (expressed inT-Cells), IFN-gamma (expressed in Th-1 cells), and IL-4 (expressed inTh-2 cells). FIG. 3 shows the levels of each cytokine as compared tonon-stimulated PBMC.

As shown in FIG. 3, ANW up-regulated IL-10 production by stimulated PMBCin a statistically significant way (T test: p<0.05). Interleukin IL-10is an important immunoregulatory cytokine. Its main biological functionis to limit and terminate inflammatory responses, and to regulate thedifferentiation and proliferation of several immune cells. IL-10deficiency is regarded as pathophysiologically relevant in inflammatorydisorders characterized by a type 1 cytokine pattern such as psoriasis.Thus, the immune-modulating properties of ANW and, specifically, theIL-10 activating properties of ANW, suggest than ANW can be used as adirect therapeutic agent for several skin diseases.

Example 5 Objective

Compare the stability of acidic nanoclustered water (ANW) with thereported stability of other electrolyzed waters of similar pH, ORP, andcomposition.

Comparison of ANW with Electrolyzed Oxidizing (EO) Water of Soo-Voo Len

The stability of ANW in open and closed conditions at 25° C. wascompared with the stability of EO water as reported in the article“Effects of Storage Conditions and pH on Chlorine Loss in ElectrolyzedOxidizing (EO) Water”—Journal of Agricultural and Food Chemistry—2002,50, 209-212 by Soo-Voon Len, et al.

In Soo-Voo Len, electrolyzed water with an acidic pH (2.5-2.6),OPR >1000 mV (1020-1120), and a free chlorine content ˜50 ppm (53-56ppm) was generated with an ROX-20TA device manufactured by HoshizakiElectric Inc. (Aichi, Japan) using a current intensity of 14 Ampere and7.4 Volt. The article reports that in an open condition at 25° C., thechlorine in the electrolyzed water was completely lost after 30 hourswhen agitated, and after 100 hours when not agitated. Furthermore, asseen in FIG. 1 of the article, the free chlorine was almost completelylost after 10 hours in open, agitated, diffused light conditions. Thearticle also reports that in a closed dark condition at 25° C., the freechlorine in the electrolyzed water decreased by approximately 40% after1400 hours (about 2 months).

In comparison, ANW stored in an open condition at 25° C. withoutagitation completely lost chlorine after 240 hours (10 days), more thantwice as long as the EO water in Soo-Voo Len (100 hours). Furthermore,ANW stored in an open condition at 25° C. with agitation and lightcompletely lost chlorine after 24 hours, more than twice as long as theEO water in Soo-Voo Len (10 hours). Finally, ANW stored in a closedcondition at 25° C. lost 8.44% of free chlorine after 3 months, lessthan a quarter as much as was lost from the EO water in Soo-Voo Lenafter about 2 months (40%).

Comparison of ANW with Electrolyzed Oxidizing (EO) Water of Shun-Yao Hsu

The stability of ANW in closed conditions at around 30° C. was comparedwith the stability of EO water as reported in the article “Effects ofstorage conditions on chemical and physical properties of electrolyzedoxidizing water”—Journal of Food Engineering 65 (2004) 465-471 byShun-Yao Hsu, et al.

In Shun-Yao Hsu, the electrolyzed water of “formulation J” had an acidicpH (2.61), OPR=1147 mV, and a free chlorine content of 56 ppm. Thearticle reports that in a closed condition at 25-30° C., the freechlorine in the electrolyzed water was 43 ppm after 21 days, a 23% loss.In comparison, ANW samples stored in closed conditions at 25° C., 30°C., and 40° C. without agitation lost 8.44%, 8.64%, and 15.43% of freechlorine after 3 months and 12.14%, 19.13%, and 18.31% of free chlorineafter 1 year, respectively.

Example 6

The properties and composition of Acidic Nanoclustered Water were testedand found to have specifications as reported below in Table 11. Theproperties and composition Acidic Nanoclustered Water were also analyzedas reported below in Table 12.

TABLE 11 Acid Water Specifications Test Item Method SpecificationAppearance Naked eye Liquid Odour Smell Characteristic Colour Naked eyeColourless pH as is @ 25° C. <3.00 by Mettler Toledo pHmeter SevenMulti-Potentiometric Determination (Ph Eur. 2.2.3 - Current Ed.)OxidoReductive as is @ 25° C. >1100.0 Potential by Mettler Toledocombination redox electrode (P/N ORP (mV) 51343200) PotentiometricTritation (Ph Eur. 2.2.20 - Current Ed.) Conductivity as is @ 25° C.<1300 (uS cm⁻¹) Free Chlorine Internal Method M37-07 40.0-70.0 Assay(mg/l or Spectrophotometric Method ppm) source APAT IRSA CNR HandBookVolume 2 - Ref 4080 Total Chlorine Internal Method M37-07 40.0-70.0Assay (mg/l or Spectrophotometric Method ppm) source APAT IRSA CNRHandBook Volume 2 - Ref 4080 Total Chlorine Internal Method M37-0740.0-70.0 Assay (mg/l or Iodometric Method ppm) source APAT IRSA CNRHandBook Volume 2 - Ref 4080 Chloride Assay Internal Method M05-08<200.0 ((mg/l or ppm) Spectrophotometric Method source APAT IRSA CNRHandBook Volume 2 - Ref 4090 Chlorites EPA 300.1, 1997 (as ClO₂) <100(μg/l or ppb)) Chlorates EPA 300.1, 1997 <1 (mg/l or ppm) ¹⁷O-NMR (Hz)¹⁷O-NMR Spectrometer <50 Linewidth @ 50% Heavy metals ICP Method  <10ppm [Ag, As, Bi, Cd, Cu, Hg, Mo, Pb, Sb, Sn] Yttrium by EPA 200.8 1994(0.1 μg/l detection limit) <0.1 ppm (ICP Method) Zinc by EPA 200.8 1994(0.1 μg/l detection limit) <0.1 ppm (ICP Method) Iridium by EPA 200.81994 (0.1 μg/l detection limit) <0.1 ppm (ICP Method) Titanium by EPA200.8 1994 (0.1 μg/l detection limit) <0.1 ppm (ICP Method) Zirconium byEPA 200.8 1994 (0.1 μg/l detection limit) <0.1 ppm (ICP Method)Ruthenium by EPA 200.8 1994 (0.1 μg/l detection limit) <0.1 ppm (ICPMethod)

TABLE 12 Acid Water Test Results ANW ANW ANW LOT LOT LOT LCOVI/57 LCOX/5LCOX/1 Appearance colourless Same Same liquid with light chlorine smell(like swimming pool) Free Chlorine Assay (mg/l) 53.1 48.6 49.9Spectrophotometric Method Total Chlorine Assay (mg/l) 52.1 48.6 49.0Spectrophotometric Method Total Chlorine Assay (mg/l) 60.6 54.9 56.7Iodometric Method Chloride Assay (mg/l) 138 194.0 183.4 UNI 24012(Mercurimetric method) Chlorites μg/l (as ClO₂) by <100 100 <100 EPA300.1 1997 (detection limit 100 μg/l) Chlorates mg/l by EPA 1.20 1.5 0.9300.1 1997 (detection limit 0.1 mg/l) pH 2.59 2.71 2.81 (as is byMettler Toledo pH meter Met Rohm 744) ORP by Mettler Toledo 1151.81121.7 1110.5 PT4805-60-88TE-S7/120 combination redo electrode ¹⁷O NMR(Linewidth @ 45.76 45.33 46.07 50%-Hz) Heavy Metals <10 ppm <10 ppm <10ppm (Ag, As, Bi, Cd, Cu, Hg, Mo, Pb, Sb, Sn)

Example 6

The stability of Acidic Nanoclustered Water compositions containingdifferent amounts of chloride ion were tested both in storage and in theopen air. The low chloride composition contained less than 200 ppmchloride, and the high chloride composition contained 1100 ppm chloride.

To test storage stability, the compositions were stored in a closedcondition at 25° C. and 60% relative humidity, and were not agitated orexposed to diffused light. After 3 and 12 months, the low chloridecomposition had a loss of free chlorine of 8.44% and 12.14%,respectively. In contrast, the high chloride composition had a loss offree chlorine of 27.4% after only 3 months.

To test open air stability, the two compositions were kept open,agitated, and exposed to light for 24 hrs at a temperature of 30° C. Asillustrated in FIG. 5, the high chloride composition lost free chlorineat a greater rate than the low chloride composition.

These results demonstrate that the stability of ANW is dependent ofchlorine remaining HClO, which prevents both evaporation anddecomposition.

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All references cited herein, including patents, patent applications, andpublished patent applications, are hereby incorporated by reference intheir entireties, whether or not each is further individuallyincorporated by reference.

1) A method of treating a condition selected from cataract, ocularkeratitis, corneal neovascularization, epithelium deficiency, andchronic opacity in a lens in an animal patient in need thereof,comprising topically administering to an eye of said patient acomposition comprising a therapeutically effective amount of a high ORPacidic water. 2) The method of claim 1, wherein said high ORP acidicwater has an ORP of greater than 1100 mV, and a pH of from 0.5 to 5.0.3) The method of claim 1, wherein said high ORP acidic water has a NMRhalf line width using ¹⁷O of from 42 to 60 Hz, and a pH of about 0.5 toabout 5.0. 4) The method of claim 1, wherein said administering stepcomprises dropping said composition directly into said eye. 5) Themethod of claim 1, wherein said administering step comprises applyingsaid composition to said eye with a piece of gauze. 6) The method ofclaim 1, wherein said administering step comprises applying saidcomposition to said eye 1 to 20 times per day, for at least 14 days. 7)An electrolytic acid water comprising free chlorine, wherein: a) from95% to 99.9% of said free chlorine is present in the form ofhypochlorous acid; b) said water has a pH of from 1.0 to 4.0; and c)said water has an ORP of greater than 1100 mV. 8) An electrolytic acidwater comprising free chlorine, wherein: a) from 90% to 99.9% of saidfree chlorine is present in the form of hypochlorous acid; b) said waterhas a pH of from 1.0 to 4.0; and c) said water has an ORP of greaterthan 1100 mV. 9) The electrolytic acid water of claim 7, wherein therelative amount of HOCl and Cl₂ is from 99.9% HOCl and 0.1% Cl₂ to 95%HOCl to 5% Cl₂. 10) The electrolytic acid water of claim 7, wherein therelative amount of HOCl and Cl₂ is from 99.5% HOCl and 0.5% Cl₂ to 98.5%HOCl to 1.5% Cl₂. 11) The electrolytic acid water of claim 7, having aNMR half line width using ¹⁷O of from 42 to 60 Hz. 12) The electrolyticacid water of claim 7, wherein said water has a conductivity of from1200 to 1400 uS/cm. 13) The electrolytic acid water of claim 7, whereinsaid water maintains 90% of said free chlorine after a storage period of3 months at room temperature. 14) The electrolytic acid water of claim7, wherein said water maintains 80% of said free chlorine after astorage period of 3 months at room temperature.